Role of ABH1's AP endonuclease activity in immunoglobulin gene diversification: The focus of this proposal is to determine whether ABH1 (mammalian homologue I of the Escherichia coli AlkB DNA repair enzyme) functions in lymphocytes to cleave abasic sites and generate breaks that are the requisite intermediate for class switch recombination (CSR) and gene conversion (GC) and that may promote certain types of somatic hypermutations (SH) during B cell affinity maturation. These processes are known to be initiated by activation induced cytidine deaminase (AID) acting on single-stranded DNA in V or switch regions. Uracil N-glycosylase (Ung) acts on the deaminated cytidine (now a uracil) to generate an abasic site. Our recent biochemical studies of ABH1 revealed it to possess an endonucleolytic activity with specificity for cleaving at abasic sites in double-stranded DNA. No direct connection has been reported between ABH1 and CSR, GC, or SHM, but no evidence firmly implicating any known abasic site endonuclease in these processes has been forthcoming. If ABH1 is the enzyme responsible for cleaving AID/Ung induced abasic sites in developing B lymphocytes, this study will have high impact for understanding several critical steps in B cell differentiation, substantially advancing progress in the field. The specific goals of the proposed research are to: 1. Create ABH1-/- mice from ABH1+/flox mice. 2. Determine whether CSR proceeds normally in B cells from ABH1-/- mice. 3. Determine whether SHM is altered in B cells from ABH1-/- mice. PUBLIC HEALTH RELEVANCE: Survival of higher vertebrates in normal environments requires a highly adaptive immune system to generate an immense repertoire of distinct antigen receptors. In particular, activation-induced deaminase (AID) and uracil DNA N-glycosylase (Ung) create abasic sites in DNA as required steps of class switch recombination, gene conversion, and somatic hypermutation. The studies described in this proposal will identify whether ABH1, the human homologue of the Escherichia coli AlkB DNA repair enzyme, uses its newly-detected ability to cleave abasic sites of DNA to facilitate immunoglobulin gene diversification system in B cells.